大鼠骨髓间充质干细胞移植对小鼠实验性自身免疫性脑脊髓炎治疗作用的初步探究

发布时间：2018-03-24 00:08

本文选题：骨髓间充质干细胞　切入点：异种移植　出处：《贵州医科大学》2017年硕士论文

【摘要】：目的:体外分离培养鉴定大鼠骨髓间充质干细胞(Rat bone marrow mesenchymal stem cells,Rat BM-MSCs)并初步探究其对小鼠实验性自身免疫性脑脊髓炎(experimental autoimmune encephalomyelitis,EAE)的治疗作用。方法:1.取SPF级大鼠骨髓,贴壁培养法获得BM-MSCs,观察细胞形态、对P3代细胞进行细胞表面抗原标志物CD29、CD90、CD106、CD45鉴定以及BM-MSCs成骨、成脂分化鉴定。2.取20只雌性C57BL/6小鼠,随机分为三组:正常对照组(Control组)、PBS组和Rat BM-MSCs组。Rat BM-MSCs细胞移植后小鼠作为Rat BM-MSCs组,PBS移植后小鼠作为PBS组,未作处理小鼠作为正常对照组。MOG35-55联合完全弗氏佐剂充分乳化后,背部皮下多点注射免疫,诱发小鼠EAE。3.小鼠免疫后第38d腹腔注射Rat BM-MSCs(1×106cells/200μl)进行治疗,PBS组注射等体积PBS,10d后再次治疗,每天进行神经功能评分观察各组小鼠神经功能变化。4.二次移植治疗12d后取各组小鼠脊髓:HE染色及Luxol Fast Blue染色观察脊髓炎性细胞浸润以及髓鞘脱失情况;5.Rat BM-MSCs细胞二次移植后第12d获取各组小鼠脾脏制成单细胞悬液,脾细胞CFSE标记后10μg/ml ConA和MOG35-55刺激培养3d,收细胞,流式细胞术检测脾细胞增殖情况;6.Rat BM-MSCs细胞二次移植后第12d收集小鼠外周血血清,Ellisa技术检测正常对照组、PBS组和Rat BM-MSCs组外周血中细胞因子:干扰素γ(IFN-γ),白介素17(IL-17)的含量。结果:1.Rat BM-MSCs成功分离培养,P3代细胞呈梭形或多角形,呈典型旋涡状生长;流式细胞术显示Rat BM-MSC P3代细胞表达抗原CD29、CD90、CD106,不表达CD45;BM-MSCs体外诱导能向成骨及成脂方向分化。2.C57BL/6小鼠发病后,EAE小鼠神经功能症状加重,临床评分逐渐升高,BM-MSCs移植后,Rat BM-MSCs组小鼠神经功能缺损症状较PBS组减轻,评分降低,二次移植后临床症状维持进一步好转。3.HE染色结果显示,Rat BM-MSCs组脊髓炎性细胞浸润比同时间点PBS组减轻(P0.05);Luxol Fast Blue染色结果显示,Rat BM-MSCs组脱髓鞘比同时间点PBS组减轻(P0.05);病理结果分析,BM-MSCs移植后小鼠脊髓炎症浸润和脱髓鞘情况得以改善。4.流式细胞术分析,ConA和MOG35-55刺激培养3d后,PBS组和Rat BM-MSCs组脾细胞增殖明显增加,而Rat BM-MSCs组又较PBS组降低。5.ELISA结果分析,与正常对照组相比,PBS组小鼠外周血血浆中促炎细胞因子IFN-γ、IL-17含量升高;与PBS组相比,Rat BM-MSCs组小鼠外周血血浆促炎细胞因子IFN-γ、IL-17含量降低(P0.05)。结论:全骨髓贴壁培养法能有效分离纯化BM-MSCs,大鼠BM-MSCs移植对小鼠EAE模型有治疗作用,且其治疗机制可能与抑制EAE小鼠体内炎性T细胞增殖和调节细胞因子的分泌有关。
[Abstract]:Objective: to isolate and identify rat bone marrow mesenchymal stem cells (BM-MSCs) in vitro and to explore the therapeutic effect of BM-MSCs on experimental autoimmune autoimmune encephalomyelitis (EAE) in mice. BM-MSCswere obtained by adherent culture method. The morphology of P3 cells was observed. CD29, CD90, CD106, CD45, BM-MSCs osteogenesis, adipogenic differentiation, and 20 female C57BL/6 mice were identified. The mice were randomly divided into three groups: normal control group (control group) and Rat BM-MSCs group. The mice were treated as PBS group after Rat BM-MSCs transplantation, and the mice were emulsified with full Freund's adjuvant and without treatment as normal control group (.MOG35-55) and complete Freund's adjuvant (Freund's adjuvant). Rat BM-MSCs(1 脳 106cells/200 渭 l was injected intraperitoneally on the 38th day after immunization to induce mice to be immunized by subcutaneous multi-point injection on the back. The mice in the PBS group were treated again after 10 days of iso-volume PBSs injection. After 12 days of second transplantation, the spinal cord of each group was stained with Luxol Fast Blue and Luxol Fast HE staining to observe the infiltration of inflammatory cells in spinal cord and the demyelination of myelin sheath. On the 12th day after transplantation, the spleen of each group was obtained and made into single cell suspension. Splenocytes were cultured for 3 days with 10 渭 g/ml ConA and MOG35-55 after CFSE labeling. Detection of splenocyte proliferation by flow cytometry 6. The levels of cytokines (IFN- 纬, IL-17IL-17) in peripheral blood of mice in normal control group and Rat BM-MSCs group were detected by flow cytometry on the 12th day after secondary transplantation of BM-MSCs cells. Results: 1.Rat BM-MSCs successfully isolated and cultured pP3 cells in fusiform or polygonal shape. Flow cytometry showed that Rat BM-MSC P3 cells expressed antigen CD29, CD90, CD106, and CD45 BM-MSCs could induce osteogenesis and adipogenic differentiation in vitro. The clinical scores of BM-MSCs were gradually increased, and the neurological impairment symptoms in the BM-MSCs group were decreased compared with those in the PBS group, and the scores of the BM-MSCs were lower than those of the PBS group. The results of HE staining showed that the infiltration of inflammatory cells in spinal cord in BM-MSCs group was lighter than that in PBS group at the same time point. The results of Fast Blue staining showed that the demyelination of BM-MSCs group was lower than that of PBS group at the same time point, and the pathological changes of BM-MSCs group was lower than that of PBS group at the same time point. Results the inflammatory infiltration and demyelination of spinal cord were improved after transplantation of BM-MSCs. The proliferation of splenocytes was significantly increased in Rat BM-MSCs group and Rat BM-MSCs group after 3 days of culture stimulated by Cona and MOG35-55 by flow cytometry. Compared with the normal control group, the plasma pro-inflammatory cytokine IFN- 纬 IL-17 in the Rat BM-MSCs group was higher than that in the normal control group. Compared with PBS group, the plasma pro-inflammatory cytokine IFN- 纬 IL-17 in BM-MSCs group decreased P0.05.Conclusion: the whole bone marrow adherent culture method can effectively isolate and purify BM-MSCs. Rat BM-MSCs transplantation has therapeutic effect on mouse EAE model. The therapeutic mechanism may be related to the inhibition of inflammatory T cell proliferation and regulation of cytokine secretion in EAE mice.
【学位授予单位】：贵州医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R744.51

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